The goal of this project is to significantly impact biology and medicine by establishing gene targeting technology in the rat using spermatogonial stem cells given pluripotent rat ES cells have not been obtained. The rat represents the most widely used laboratory-scale mammalian model for human physiology and disease. However, despite its wide popularity in science, technology to produce rats with targeted genomic modifications has yet to be established. Therefore, one long-term goal of this application is to develop technology that will allow scientists to use spermatogonial stem cells to produce transgenic rat models with specific genomic modifications; such animal models will represent alternatives to the mouse in terms of answering important health-related questions. A second long term goal of this application will be to use the generated technology to unravel the molecular biology, physiology and pluripotent nature of spermatogonial stem cells. To realize these long-term goals, the immediate goals in this proposal are to genetically modify rat spermatogonial stem cells in culture at defined genomic sites by using Cre-lox (Specific Aim 1) and homologous recombination (Specific Aim 2) technology. The modified stem cells will be expanded in culture using positive and negative selectable markers. The selected lines will be transplanted into rat testes where they will develop into transgenic spermatozoa and then used for germline transmission of the targeted rat alleles through natural mating. Due to the popularity of the laboratory rat in science, animal models generated using sperm stem cells by the proposed technology will have a positive impact on public health issues by greatly facilitating research in many different medical fields. This would include studies in the rat on neurological and sensory disorders, drug addiction, cardiovascular and renal disease, immune disease, gastrointestinal disease, pulmonary disorders, nutrition, aging, reproduction and cell based therapies. Due to the popularity of the laboratory rat in science, animal models generated using sperm stem cells by the proposed technology will have a positive impact on public health issues by greatly facilitating research in many different medical fields. This would include studies in the rat on neurological and sensory disorders, drug addiction, cardiovascular and renal disease, immune disease, gastrointestinal disease, pulmonary disorders, nutrition, aging, reproduction and cell based therapies. [unreadable] [unreadable] [unreadable]